It is crucial to have an accurate antimicrobial resistance profile to design effective treatment plans for multidrug-resistant tuberculosis (MDR-TB). Despite widespread clinical use, accurate testing procedures for para-aminosalicylic acid (PAS) susceptibility testing procedures have not been established. For a study, researchers intended to determine the minimum detectable concentration of PAS in the Mycobacterial Growth Indicator Tube (MGIT) 960. In all, 102 clinical isolates were analyzed, 82 of which were wild-type and 20 of which were resistotype. MGIT 960 was used to evaluate the MIC (minimum inhibitory concentration). Investigators employed whole-genome sequencing to find mutations that might make the bacteria resistant to PAS.

The possible mechanism of drug resistance of the folC mutant was analyzed by doing sequence alignment and structural modeling. In general, there was a clear demarcation between wild-type and resistotype isolates in the MIC distribution. The wild-type population was all at least 1 dilution below 4 μg/ml, and the resistotype population was no lower than 4 μg/ml, indicating that 4 μg/ml was an appropriate critical concentration to separate these 2 populations. About 12(20%) of the mutant isolates were thyA mutants,  2(10%) were dfrA upstream mutants, and the remaining mutant isolates were folC mutants. Mutations in thyA and folC occurred randomly throughout the entire gene.

All thyA mutations conferred high-level resistance to PAS (MIC > 32 μg/ml), whereas folC mutations resulted in a much wider range of MICs (4 to > 32 μg/ml), with sequence and structural analysis providing some insight into the likely causes of this variation. Study group suggests a provisional critical concentration of 4 μg/ml for MGIT 960. Changes in the genes thyA and folC in MTB isolates are the primary cause of their resistance to PAS. High levels of resistance to PAS can be achieved by deleting the thyA locus entirely. PCR-based mutation study for PAS susceptibility is complicated by the widespread distribution of many different mutations across the entire full-length folC gene.